Protein NMR Sample Requirements

Although the NMR facility is focused on small molecule and solid state NMR, biomolecular research is a major emphasis. Biomacromolecular solution NMR study is the most important focus. Listed below are some basic common requirements for sample preparation, in particular protein samples, of solution biomolecular NMR experiments. For more information on sample preparation of membrane protein studies with either solution or solid-state NMR techniques please contact our expertise and refer to relevant references.

• Common Profile: 0.1 - 2.5 mM concentration, typically about 1 mM; stable at room temperature over weeks;²H/¹³C/¹⁵N isotope labeling; mono-disperse; purity greater than 95%; neither paramagnetic impurities nor solid particles present; organic buffer components in perdeuterated forms; 10 - 50 µM DSS or TSP as inner reference; 90% - 95%H₂O:10% - 5%D₂O.
• Volume: 500 - 550 µL for regular 5 mm NMR tubes and 260 - 300 µL for Shigemi tubes; 2 - 30 mg of protein of 8 - 30 kDa molecular weight, typically about 10 mg.
• Stability: No precipitation in proper buffers; no degradation with removal of proteases or addition of protease inhibitors; no decomposition by bacteria with addition of 10 - 50 µM NaN₃ or use of micro-filtration; no oxidation (SH-groups) through addition of DTT.
• Buffer: pH: neutral or slightly acidic; phosphate buffer preferred; away from protein pI value. Ionic strength: less than 500 mM for conventional probes and less than 100 mM for cryogenic probes.
• Overexpression: E. coli strains preferred; chemically defined media (e.g. M₉ and modifications) with ¹³C isotope enriched glucose/glycerol and ¹⁵N isotope enriched NH₄Cl/(NH₄)₂SO₄ as sole carbon source and nitrogen source, respectively, for uniformly ¹³C/¹⁵N labeling; protein yield more than 5 mg/L preferred.